Synaptic plasticity at glutamatergic synapses is critical for adaptive learning and conditioned responding to opiates, heroin and morphine, and other positive reinforcers. This plasticity is attributed, in part to changes in the subcellular distribution of ionotropic (AMPA and/or NMDA) glutamate receptor subunits. Metabotropic glutamate (mGlu) receptors and endocannabinoids, active at the cannabinoid type-1 (CB1) receptor, are increasingly implicated in the synaptic plasticity associated with opiate-induced conditioned place preference (CPP) and locomotor activation, presumably a reflection of drug craving. These opiate-induced behavioral adaptations are dependent on mu-opioid receptors in the ventral tegmental area (VTA), but are also influenced by activation of mu-opioid receptors in the nucleus accumbens (Acb) and other limbic brain regions that receive input from dopamine neurons in the VTA. The opiate-induced disinhibition of VTA mesolimbic dopamine neurons results in dopamine release and activation of dopamine D1- and D2-like receptors. The dopamine D2-like receptors in the Acb are of special interest, because of their unique capacity to not only modulate glutamatergic transmission and release of preference-associated enkephalin- type opioid peptides, but also to inhibit the release of dopamine and to form functional heterodimers with the CB1 receptor. Thus, the location of multiple G-protein coupled (CB1, mGluR, mu-opioid, and dopamine D2) receptors identifies those neurons within the VTA and Acb that are most likely to show long-lasting and possibly opposing changes in the strength of glutamatergic synapses that correlate with the behavioral adaptations to opiates. High resolution electron microscopic immunolabeling is one of the few methods capable of revealing small, but functionally significant, in vivo changes in the surface/synaptic distribution of ionotropic glutamate receptor subunits. This method, behavioral testing, and genetic manipulations will be used to test the Central Hypothesis that morphine-induced behavioral adaptations are correlated with CB1 receptor-dependent plasticity at glutamatergic synapses in VTA and/or Acb neurons of distinct transmitter and GPCR phenotypes. We will determine in each region whether (1) key GPCRs have subcellular locations consistent with their involvement in morphine-induced plasticity at glutamatergic synapses, (2) repeated, intermittent, morphine administration produces a behaviorally correlated change in the subcellular distribution of the essential NMDA NR1 and AMPA receptor subunits that could enable distinct forms of synaptic plasticity in neurons of chemically and functionally opposed phenotypes, and (3) CB1 gene deletion affecting CPP and locomotor activity produces changes in opioid and dopamine systems, and in synaptic plasticity occurring at glutamatergic synapses. The results will have many important implications for understanding the cross-sensitization between opiates and cannabinoids and for devising new treatments for addictive diseases. The opiates, heroin and morphine, are highly addictive substances that produce long-lasting changes in the brain's reward (mesolimbic dopamine) circuit, which increase craving and demand repeated use. Endocannabinoids, the brain's own marihuana, are key modulators within this circuit, and play an as yet poorly understood role in the adaptive behavioral response to morphine. To better understand and treat human addictive diseases, this grant application proposes to use high resolution electron microscopy, genetic manipulations, and behavioral testing in rodents to determine how endocannabinoids and opioids interactively control the communication between nerve cells in the reward circuit.